基于植物干细胞培养体系的长春新碱生物合成途径及其代谢调控机理研究

Vincristine (VCR) is derived from Catharanthus roseus; however, it is currently extremely expensive because of its low production and supply. Although chemical synthesis of VCR has been done, commercialization of this method is still limited due to the high cost. Additionally, production of VCR by genetic engineering is unavailable because VCR biosynthetic pathway remains unclear. Generally, one of best ways to investigate biosynthetic pathways is by plant cell culture systems; however, vindoline, a key intermediate in VCR biosynthetic pathway, cannot be produced by dedifferentiated cell systems. Therefore, it is of much importance to identify a novel plant cell culture system that enables us to elucidate VCR biosynthetic pathway and the mechanism of its metabolic regulation. Plant stem cells are homogenous undifferentiated cells that still retain all of the genetic information of parental plant cells, and their genetic traits are more stable than those of callus. Recently, we have successfully established the stem cell culture system of C. roseus and were able to detect the production of vindoline and VCR in this system. The present project aims to further optimize the stem cell culture system of C. roseus, to isolate and identify intermediates in VCR biosynthetic pathway by chromatographic and spectroscopic techniques, to isolate and purify key enzymes in this biosynthetic pathway, and to determine the amino acid sequence of these enzymes by protein sequencing techniques. This project will for the first time investigate VCR biosynthetic pathway by the plant stem cell culture system. As a result, this project will provide crucial scientific basis needed to mass-produce vincristine by the plant stem cell system and advance related fields of biotechnology.

长春新碱(VCR)源自长春花，因植物含量低、化学合成无法产业化、生物合成途径不清楚而致基因工程法难以实现，故VCR原料短缺、价格奇高。植物细胞体系法是研究生物合成途径最佳方法，但常用的脱分化细胞体系却不能合成VCR的关键中间体文多灵。故寻找新型植物细胞培养体系，以阐明VCR生物合成途径及其代谢调控机理意义重大。植物干细胞体系为均一的未分化细胞，保留了母本细胞的全套遗传信息且遗传性状比脱分化细胞更为稳定。我们前期研究结果显示，在构建的长春花干细胞培养体系中检测到文多灵和VCR的生成。本项目拟在此基础上，进一步优化长春花干细胞培养体系；运用色谱和光谱技术分离和鉴定中间体；分离纯化VCR生物合成过程中的关键酶，利用谱学和蛋白质测序技术研究其氨基酸序列。此项目系国内外首次利用植物干细胞体系法研究天然药物化学成分生物合成途径，研究结果可为植物干细胞新用途和生物技术方法解决VCR原料来源提供理论基础。

本项目以长春花(Catharanthuss roseus)干细胞培养体系，即形成层分生组织细胞(cambial meristematic cells, CMCs)为研究对象，对长春花CMCs的生理生化特性进行系统的研究，使用多种策略促进萜类吲哚生物碱(terpenoid indole alkaloids, TIAs)生物合成，并对其代谢调控进行研究。本论文首次对长春花CMCs培养体系进行了较系统的研究，主要得到如下结果：(1)与DDCs相比，CMCs具有典型的干细胞特征(如小而多的液泡、对博来霉素敏感性高)和多种优良特性，如可合成文多灵、长春质碱、阿玛碱和长春碱等多种生物碱，长时间培养后仍保持其生长和生物碱合成的稳定性等；(2)对于体外培养的长春花CMCs，使用如下两种方法可有效提高TIAs的含量，第一种是使用复合诱导子(β-CD/ MeJA)和生物反应器，第二种是使用酶抑制剂(洛伐他汀和萘普生)阻断旁路代谢途径；(3)文多灵、长春质碱和阿玛碱合成的前体IPP来源于MEP途径，与MVA途径无关。综上，长春花CMCs为生产重要活性TIAs提供了一个经济的、环境友好的来源，并为TIAs的代谢调控研究提供了一个新的工具。此外，CMCs可能为植物干细胞的研究提供一个新的生物学工具。

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